Methods and Compositions for Inhibiting Akt3

ABSTRACT

Compounds and compositions for selectively inhibiting Akt3 are provided. Methods of using the compounds are also provided. Because Akt3 modulates the suppressive function of natural Tregs and the polarization of induced Tregs, the disclosed compounds are useful for modulating immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. ProvisionalPatent Application No. 62/279,150 filed on Jan. 15, 2016, and which isincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jan. 17, 2017, as a text file named“017_ST25.txt” created on Jan. 17, 2017, and having a size of 11 Kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The invention is generally directed to compositions and methods forselective inhibition of Akt3 activity, and methods of use thereof formodulating regulator T cells.

BACKGROUND OF THE INVENTION

Regulatory T cells (Tregs) are a subset of CD4+ T cells that suppressimmune responses and are essential mediators of self-tolerance andimmune homeostasis (Sakaguchi, et al., Cell, 133, 775-787 (2008)).Depletion or inactivation of Tregs results in the development of severeautoimmunity (Sakaguchi, et al., J. Immunol., 155, 1151-1164 (1995)),and their accumulation inhibits anti-tumor immunity (Dannull, et al.,The Journal of clinical investigation, 115, 3623-3633 (2005)). Tregs arecharacterized by Foxp3 expression, a transcription factor belonging tothe Forkehead Box family of transcription factors. The Foxp3 is a masterregulator of Tregs, as it is necessary for their development andfunction (Hori, Science, 299, 1057-1061 (2003); Fontenot, et al., NatImmunol., 4(4):330-6 (2003). Epub 2003 Mar. 3; Khattri, et al., NatImmunol., 4(4):337-42 (2003). Epub 2003 Mar. 3)).

There are two major types of Tregs: thymus-derived Tregs (or naturalTregs (nTregs)) that constitute 5-10% of the total peripheral CD4+ Tcells, and peripheral TGFβ-induced Tregs (iTregs). Both types are shownto have immunosuppressive properties mediated via several processes thatinvolve immunosuppressive soluble factors or cell contact (Bluestone, etal., Nat Rev Immunol, 3, 253-257 (2003); Glisic, et al., Cell and TissueResearch, 339, 585-595 (2010); Hori, Science, 299, 1057-1061 (2003);Sakaguchi, Cell, 101, 455-458 (2000); Sakagushi, et al., Curr. TopMicrobiol. Immunol., 305, 51-66 (2006); Sakagushi, et al., Immunol.,Rev., 212, 8-27 (2006); (Schmidt, et al., Front Immunol., 3:51 (2012)).However, the molecular mechanisms by which nTreg and iTreg develop andthen exhibit non-redundant roles to suppress the immunity are not fullyunderstood (Dipica, et al., Immunity, 35(1):109-122 (2011)).

PI3K-Akt signaling affects many processes and is central to manysignaling pathways. Akt phosphorylation and kinase activity are inducedby PI3K activation, which is, in turn, induced by several growth factorreceptors, TCR, CD28, and IL-2R, among many others (Parry, et al.,Trends in Immunology, 28, 161-168 (2007)). In mammals, there are threeAkt isoforms, namely Akt1, Akt2, and Akt3, encoded by three independentgenes. In vitro, these isoforms appear to have redundant functions, asdifferent extracellular inputs can induce similar Akt signaling patterns(Franke, Science 1, pe29-(2008)). However, isoform-specific knockoutsshow unique features and their involvement in diseases and physiologicalconditions is different (Boland, et al., American Journal of HumanGenetics, 81, 292-303 (2007); DeBosch, et al., J. Biol. Chem, 281,32841-32851 (2006); Emamian, et al., Nat Genet, 36, 131-137 (2004);Garofalo, et al., The Journal of clinical investigation, 112, 197-208(2003); George, et al., Science, 304, 1325-1328 (2004); Nakatani, etal., The Journal of Biological Chemistry, 274, 21528-21532 (1999);Tschopp, et al., Development (Cambridge, England), 132, 2943-2954(2005); Yang, et al., J. Biol. Chem., 278, 32124-32131 (2003)).

Studies have shown that Akt1 and Akt2 can negatively regulate thetranscriptional signature of Treg, thereby selectively affecting Treglineage differentiation (Sauer, et al., Proceedings of the NationalAcademy of Sciences, 105, 7797-7802 (2008a)). Additionally, although itwas shown that inhibition of Akt1 and Akt2 isoforms increase Foxp3expression in TGFβ induced iTregs (Sauer, et al., Proc. Natl. Acad. Sci.USA, 105, 7797-7802 (2008b)), the mechanism remained unclear. Anotherfinding shows that deletion of Akt2 resulted in defective iTh17 celldifferentiation but preserved nTh17 cell development (Kim, et al., NatImmunol., 14(6):611-8 (2013) Epub 2013 May 5). Further, Akt3 is alsoexpressed in immune cells and the spinal cord of Akt3 knockout mice havedecreased numbers of Foxp3+ regulatory T cells compared with wild typemice (Tsiperson, et al., J Immunol., 190(4):1528-39 (2013) Epub 2013Jan. 18)). Thus, although some studies have examined the relevance ofAkt isoform expression on T cell biology (Carson, et al., Annals of theNew York Academy of Sciences, 1103, 167-178 (2007), Crellin, et al.,Blood, 109, 2014-2022 (2007a); Crellin, et al., Journal of ImmunologicalMethods, 324, 92-104 (2007b); Haxhinasto, J. Exp. Med., 205, 565-574(2008); Li, et al., Blood, 106, 3068-3073 (2005); Patton, et al.,Biochem. Soc. Trans., 35, 167-171 (2007); Patton, et al., J. Immunology177, 6598-6602 (2006); Sauer, et al., Proc. Natl. Acad. Sci. USA, 105,7797-7802 (2008b); Walsh, et al., J. Clin. Invest., 116, 2521-2531.(2006)), the roles that Akt isoforms play in Treg function and inductionwas not clear.

Therefore, it is an object of the invention to provide compounds andcompositions for selectively inhibiting Akt3 in a subject.

It is another object of the invention to provide methods of increasing astimulatory immune response in a subject.

Still another object of the invention is to provide methods ofdecreasing a suppressive immune response in a subject.

SUMMARY OF THE INVENTION

Compounds and compositions for selectively inhibiting Akt3 are provided.Methods of using the compounds are also provided. Because Akt3 modulatesthe suppressive function of natural Tregs and the polarization ofinduced Tregs, the disclosed compounds are useful for modulating immuneresponses.

For example, methods of decreasing an immune suppressive response,increasing an immune stimulating response, or a combination thereof in asubject in need thereof are disclosed. The methods typically includeadministering the subject a composition including a compound thatselectively inhibits the bioactivity of Akt3 in an amount effective toreduce the immune suppressive response, increase the immune stimulatingresponse, or a combination thereof in the subject.

In some embodiments the immune suppressive response that is reduced isselected from the group consisting of an immune suppressive function ofnatural Treg (nTreg) and induction of conventional T cells into inducedTreg (iTreg). The immune suppressive function of nTreg can be thesecretion of one or more anti-inflammatory cytokines. Theanti-inflammatory cytokine(s) can IL10, TGFβ, or a combination thereof.

In some embodiments, the subject has cancer or an infection. Therefore,methods of treating cancers and infections by administering a subject inneed thereof an effective amount of a compound that reduces thebioavailability of Akt3 are also disclosed. Exemplary cancers that canbe treated include, but are not limited to, bladder, brain, breast,cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,pancreatic, prostate, skin, stomach, uterine, ovarian, testicular andhematologic cancers. Exemplary infectious diseases that can be treatedinclude, but are not limited to, those caused by a bacterium, virus,protozoan, helminth, or another microbial pathogen.

Exemplary compounds that selectively inhibit the activity of Akt3include compounds 1-28 described below.

Combination therapies and vaccine formulations including modulators ofAkt3 bioactivity and methods of use thereof are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an autoradiograph of an immunoblot of Tregs treated asindicated with4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamideand assayed for phosphorylation of pAkt3, pAkt1, or Actin.

FIGS. 2A-2P are histograms of FACS sorted nTregs treated as indicatedwith4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.

FIG. 3A is a schematic of a treatment regimen with4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.FIGS. 3B-3J are histograms of FACS sorted cells from mice as treated inFIG. 3A. FIG. 3K is a bar graph of MFI (CD4+FOXp3+) from animals treatedwith 5 mg/kg or 10 mg/kg4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.FIG. 3L is a bar graph of Foxp3+ Tcells-Tregs (% of CD4) of animalstreated with 5 mg/kg or 10 mg/kg of4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.

FIG. 4A is a schematic of a treatment regimen. FIGS. 4B-4J are dot plotsof flow cytometry analysis of animals treated with 5 mg/kg or 10 mg/kgof4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.FIG. 4K is a bar graph of CD4+ T cells (% of CD3) for animals treatedwith 5 mg/kg or 10 mg·kg of4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.FIG. 4L is a bar graph of CD8+ T cells (% of CD3) for animals treatedwith 5 mg/kg or 10 mg·kg of4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide.

FIG. 5A is a schematic of treatment regimen. FIG. 5B is a bar graph ofTumor volume (cm3) for from left to right, untreated, vaccine, 10 mg/kg4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide,and 10 mg/kg of4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamidewith vaccine. FIG. 5C is a bar graph of Tumor volume (cm³) for from leftto right, untreated, vaccine, 20 mg/kg4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide,and 20 mg/kg of4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamidewith vaccine. FIG. 5D is a Kaplan-Meier plot of the overall survival.

FIG. 6A is a structural diagram of compound (3) or JJ64-B. FIGS. 6B-6Gare histograms of the frequency of CD4+FoxP3+ cells treated withcompound (3) and measured by flow cytometry. FIG. 6H is a bar graph ofMFI (CD4+Foxp3) of cells treated with compound (3).

FIG. 7A is a structural diagram of compound (18). FIGS. 7B-7G arehistograms of the frequency of CD4+FoxP3+ cells from animals treatedwith compound (18) and measured by flow cytometry. FIG. 7H is a bargraph of MFI (CD4+Foxp3) of cells treated with compound (18).

FIG. 8A is a schematic diagram of a treatment regimen. FIG. 8B is a bargraph of Tumor Volume (cm³) of animals, from left to right, untreated,vaccine, 5 mg/kg compound (18), 5 mg/kg compound (18) and vaccine. FIG.8C is a bar graph of Tumor Volume (cm³) of animals, from left to right,untreated, vaccine, 10 mg/kg compound (18), 10 mg/kg compound (18) andvaccine. FIG. 8D is a Kaplan-Meier plot of the overall survival.

FIG. 9A is a structural diagram of a compound JJ64-D. FIGS. 9B-9G arehistograms of the frequency of CD4+FoxP3+ cells from animals treatedwith JJ64-D and measured by flow cytometry. FIG. 9H is a bar graph ofMFI (CD4+Foxp3) of cells treated with JJ64-D.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “stimulate expression of” means to affect expression of, forexample to induce expression or activity, or induce increased/greaterexpression or activity relative to normal, healthy controls.

The terms “immune activating response”, “activating immune response”,and “immune stimulating response” refer to a response that initiates,induces, enhances, or increases the activation or efficiency of innateor adaptive immunity. Such immune responses include, for example, thedevelopment of a beneficial humoral (antibody mediated) and/or acellular (mediated by antigen-specific T cells or their secretionproducts) response directed against a peptide in a recipient patient.Such a response can be an active response induced by administration ofimmunogen or a passive response induced by administration of antibody orprimed T-cells. A cellular immune response is elicited by thepresentation of polypeptide epitopes in association with Class I orClass II MHC molecules to activate antigen-specific CD4⁺ T helper cellsand/or CD8⁺ cytotoxic T cells. The response can also involve activationof monocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils, activation or recruitment ofneutrophils or other components of innate immunity. The presence of acell-mediated immunological response can be determined by proliferationassays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays. Therelative contributions of humoral and cellular responses to theprotective or therapeutic effect of an immunogen can be distinguished byseparately isolating antibodies and T-cells from an immunized syngeneicanimal and measuring protective or therapeutic effect in a secondsubject.

The terms “suppressive immune response” and “immune suppressiveresponse” refer to a response that reduces or prevents the activation orefficiency of innate or adaptive immunity.

The term “immune tolerance” as used herein refers to any mechanism bywhich a potentially injurious immune response is prevented, suppressed,or shifted to a non-injurious immune response (Bach, et al., N. Eng. J.Med., 347:911-920 (2002)).

The term “tolerizing vaccine” as used herein is typically anantigen-specific therapy used to attenuate autoreactive T and/or B cellresponses, while leaving global immune function intact.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “immune cell” refers to cells of the innate and acquired immunesystem including neutrophils, eosinophils, basophils, monocytes,macrophages, dendritic cells, lymphocytes including B cells, T cells,and natural killer cells.

As used herein “conventional T cells” are T lymphocytes that express anαβ T cell receptor (TCR) as well as a co-receptor CD4 or CD8.Conventional T cells are present in the peripheral blood, lymph nodes,and tissues. See, Roberts and Girardi, “Conventional and UnconventionalT Cells”, Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari andTyring (ed.)), Springer London (2008).

As used herein “unconventional T cells” are lymphocytes that express aγδ TCR and may commonly reside in an epithelial environment such as theskin, gastrointestinal tract, or genitourinary tract. Another subset ofunconventional T cells is the invariant natural killer T (NKT) cell,which has phenotypic and functional capacities of a conventional T cell,as well as features of natural killer cells (e.g., cytolytic activity).See, Roberts and Girardi, “Conventional and Unconventional T Cells”,Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari and Tyring(ed.)), Springer London (2008).

As used herein “Treg” refers to a regulatory T cell or cells. RegulatoryT cells are a subpopulation of T cells which modulate the immune system,maintain tolerance to self-antigens, abrogate autoimmune disease, andotherwise suppress immune stimulating or activating responses of othercells. Regulatory T cells come in many forms with the mostwell-understood being those that express CD4, CD25, and Foxp3.

As used herein “natural Treg” or “nTreg” refers to a regulatory T cellor cells that develop in the thymus.

As used herein “induced Treg” or “iTreg” refers to a regulatory T cellor cells that develop from mature CD4+ conventional T cells outside ofthe thymus.

The “bioactivity” of Akt3 refers to the biological function of the Akt3polypeptide. Bioactivity can be increased or reduced by increasing orreducing the activity of basal levels of polypeptide, increasing orreducing the avidity of basal levels of polypeptide, the quantity of thepolypeptide, the ratio of Akt3 relative to one or more other isoforms ofAkt (e.g., Akt1 or Akt2) of the polypeptide, increasing or reducing theexpression levels of the polypeptide (including by increasing ordecreasing mRNA expression of Akt3), or a combination thereof. Forexample, bioavailable Akt3 polypeptide is a polypeptide that has kinaseactivity and can bind to and phosphorylate a substrate of Akt3. Akt3polypeptide that is not bioavailable includes Akt3 polypeptide that ismis-localized or in-capable of binding to and phosphorylating Aktsubstrates.

As used herein, the phrase that a molecule “specifically binds” or“displays specific binding” to a target refers to a binding reactionwhich is determinative of the presence of the molecule in the presenceof a heterogeneous population of other biologics.

Under designated immunoassay conditions, a specified molecule bindspreferentially to a particular target and does not bind in a significantamount to other biologics present in the sample. Specific binding of anantibody to a target under such conditions requires the antibody beselected for its specificity to the target. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

The terms “oligonucleotide” and “polynucleotide” generally refer to anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single-and double-stranded DNA,DNA that is a mixture of single-and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. The term “nucleic acid” or“nucleic acid sequence” also encompasses a polynucleotide as definedabove.

In addition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation). The term polypeptide includes proteins and fragmentsthereof. The polypeptides can be “exogenous,” meaning that they are“heterologous,” i.e., foreign to the host cell being utilized, such ashuman polypeptide produced by a bacterial cell. Polypeptides aredisclosed herein as amino acid residue sequences. Those sequences arewritten left to right in the direction from the amino to the carboxyterminus. In accordance with standard nomenclature, amino acid residuesequences are denominated by either a three letter or a single lettercode as indicated as follows: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine(His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K),Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine(Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y),and Valine (Val, V).

“Variant” refers to a polypeptide or polynucleotide that differs from areference polypeptide or polynucleotide, but retains essentialproperties. A typical variant of a polypeptide differs in amino acidsequence from another, reference polypeptide. Generally, differences arelimited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more modifications (e.g., substitutions, additions, and/ordeletions). A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides of the disclosure and still obtain a molecule havingsimilar characteristics as the polypeptide (e.g., a conservative aminoacid substitution). For example, certain amino acids can be substitutedfor other amino acids in a sequence without appreciable loss ofactivity. Because it is the interactive capacity and nature of apolypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence and nevertheless obtain a polypeptide with likeproperties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, andcofactors. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamnine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide ofinterest.

The term “percent (%) sequence identity” is defined as the percentage ofnucleotides or amino acids in a candidate sequence that are identicalwith the nucleotides or amino acids in a reference nucleic acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For purposes herein, the % sequence identity of a given nucleotides oramino acids sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given sequence Cthat has or comprises a certain % sequence identity to, with, or againsta given sequence D) is calculated as follows:

100 times the fraction W/Z,

where W is the number of nucleotides or amino acids scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides or amino acidsin D. It will be appreciated that where the length of sequence C is notequal to the length of sequence D, the % sequence identity of C to Dwill not equal the % sequence identity of D to C.

The term “carrier” refers to an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitatethe application.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients.

The term “pharmaceutically-acceptable carrier” means one or morecompatible solid or liquid fillers, dilutants or encapsulatingsubstances which are suitable for administration to a human or othervertebrate animal.

The term “effective amount” or “therapeutically effective amount” meansa dosage sufficient to provide treatment a disorder, disease, orcondition being treated, or to otherwise provide a desired pharmacologicand/or physiologic effect. The precise dosage will vary according to avariety of factors such as subject-dependent variables (e.g., age,immune system health, etc.), the disease, and the treatment beingeffected.

The terms “individual,” “individual,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, humans, rodents, such as mice and rats, and other laboratoryanimals.

II. Compositions for Inhibiting Akt3

Compounds for selectively inhibiting Akt3 activity and methods of usethereof are disclosed. Exemplary compounds for selectively inhibitingAkt3 include the following:

and enantiomers, polymorphs, pharmaceutically acceptable salts, andderivatives thereof. As used herein, “compounds 1-28” refers to any oneor combination of 2 or more of compounds 1-28, and enantiomers,polymorphs, pharmaceutically acceptable salts and derivatives thereof.

In some embodiments, the Atk3 inhibitor is a derivative of any one ofcompounds 1-28. The term “derivative” or “derivatised” as used hereinincludes one or more chemical modifications of any one of compounds1-28, an enantiomer, polymorph, or pharmaceutically acceptable saltthereof. That is, a “derivative” may be a functional equivalent of anyone of compounds 1-28, which is capable of inducing the improvedpharmacological functional activity and/or behavioral response in agiven subject. Illustrative of such chemical modifications would bereplacement of hydrogen by a halo group, an alkyl group, an acyl groupor an amino group.

The chemical modification of any one of compounds 1-28, an enantiomer,polymorph, or pharmaceutically acceptable salt thereof may eitherenhance or reduce hydrogen bonding interaction, charge interaction,hydrophobic interaction, Van Der Waals interaction or dipole interactionbetween the compound and its target.

In some embodiments, the compound of any one of compounds 1-28 may actas a model (for example, a template) for the development of otherderivative compounds which are a functional equivalent of the compoundand which is capable of inducing the improved pharmacological functionalactivity and/or effect and/or behavioral response in a given subject.

Compounds 1-28 may be racemic compounds and/or optically active isomersthereof. In this regard, some of the compounds can have asymmetriccarbon atoms, and therefore, can exist either as racemic mixtures or asindividual optical isomers (enantiomers). Compounds described hereinthat contain a chiral center include all possible stereoisomers of thecompound, including compositions including the racemic mixture of thetwo enantiomers, as well as compositions including each enantiomerindividually, substantially free of the other enantiomer. Thus, forexample, contemplated herein is a composition including the S enantiomerof a compound substantially free of the R enantiomer, or the Renantiomer substantially free of the S enantiomer. If the named compoundincludes more than one chiral center, the scope of the presentdisclosure also includes compositions including mixtures of varyingproportions between the diastereomers, as well as compositions includingone or more diastereomers substantially free of one or more of the otherdiastereomers. By “substantially free” it is meant that the compositionincludes less than 25%, 15%, 10%, 8%, 5%, 3%, or less than 1% of theminor enantiomer or diastereomer(s).

Compounds 1-28 selectively inhibit Akt3 compared to Akt1 and Akt2. Incertain embodiments, compounds 1-28 do not inhibit Akt1 and Akt2 to astatistically significant degree. In other embodiments, inhibition ofAkt3 by compounds 1-28 is 5, 10, 15, 50, 100, 1000, or 5000 fold greaterthan their inhibition of Akt1 and Akt2.

Akt3, also referred to as RAC-gamma serine/threonine-protein kinase isan enzyme that in humans is encoded by the Akt3 gene. Akt kinases areknown to be regulators of cell signaling in response to insulin andgrowth factors and are associated with a broad range of biologicalprocesses including cell proliferation, differentiation, apoptosis,tumorigenesis, as well as glycogen synthesis and glucose uptake. Akt3has been shown to be stimulated by platelet-derived growth factor(PDGF), insulin, and insulin-like growth factor 1 (IGF1).

Akt3 kinase activity mediates serine and/or threonine phosphorylation ofa range of downstream substrates. Nucleic acid sequences for Akt3 areknown in the art. See, for example, Genbank accession no. AF124141.1:Homo sapiens protein kinase B gamma mRNA, complete cds, which isspecifically incorporated by references in its entirety, and providesthe nucleic acid sequence:

(SEQ ID NO: 1)AGGGGAGTCATCATGAGCGATGTTACCATTGTGAAGGAAGGTTGGGTTCAGAAGAGGGGAGAATATATAAAAAACTGGAGGCCAAGATACTTCCTTTTGAAGACAGATGGCTCATTCATAGGATATAAAGAGAAACCTCAAGATGTGGATTTACCTTATCCCCTCAACAACTTTTCAGTGGCAAAATGCCAGTTAATGAAAACAGAACGACCAAAGCCAAACACATTTATAATCAGATGTCTCCAGTGGACTACTGTTATAGAGAGAACATTTCATGTAGATACTCCAGAGGAAAGGGAAGAATGGACAGAAGCTATCCAGGCTGTAGCAGACAGACTGCAGAGGCAAGAAGAGGAGAGAATGAATTGTAGTCCAACTTCACAAATTGATAATATAGGAGAGGAAGAGATGGATGCCTCTACAACCCATCATAAAAGAAAGACAATGAATGATTTTGACTATTTGAAACTACTAGGTAAAGGCACTTTTGGGAAAGTTATTTTGGTTCGAGAGAAGGCAAGTGGAAAATACTATGCTATGAAGATTCTGAAGAAAGAAGTCATTATTGCAAAGGATGAAGTGGCACACACTCTAACTGAAAGCAGAGTATTAAAGAACACTAGACATCCCTTTTTAACATCCTTGAAATATTCCTTCCAGACAAAAGACCGTTTGTGTTTTGTGATGGAATATGTTAATGGGGGCGAGCTGTTTTTCCATTTGTCGAGAGAGCGGGTGTTCTCTGAGGACCGCACACGTTTCTATGGTGCAGAAATTGTCTCTGCCTTGGACTATCTACATTCCGGAAAGATTGTGTACCGTGATCTCAAGTTGGAGAATCTAATGCTGGACAAAGATGGCCACATAAAAATTACAGATTTTGGACTTTGCAAAGAAGGGATCACAGATGCAGCCACCATGAAGACATTCTGTGGCACTCCAGAATATCTGGCACCAGAGGTGTTAGAAGATAATGACTATGGCCGAGCAGTAGACTGGTGGGGCCTAGGGGTTGTCATGTATGAAATGATGTGTGGGAGGTTACCTTTCTACAACCAGGACCATGAGAAACTTTTTGAATTAATATTAATGGAAGACATTAAATTTCCTCGAACACTCTCTTCAGATGCAAAATCATTGCTTTCAGGGCTCTTGATAAAGGATCCAAATAAACGCCTTGGTGGAGGACCAGATGATGCAAAAGAAATTATGAGACACAGTTTCTTCTCTGGAGTAAACTGGCAAGATGTATATGATAAAAAGCTTGTACCTCCTTTTAAACCTCAAGTAACATCTGAGACAGATACTAGATATTTTGATGAAGAATTTACAGCTCAGACTATTACAATAACACCACCTGAAAAATATGATGAGGATGGTATGGACTGCATGGACAATGAGAGGCGGCCGCATTTCCCTCAATTTTCCTACTCTGCAAGTGGACGAGAATAAGTCTCTTTCATTCTGCTACTTCACTGTCATCTTCAATTTATTACTGAAAATGATTCCTGGACATCACCAGTCCTAGCTCTTACACATAGCAGGGGCACCTTCCGACATCCCAGACCAGCCAAGGGTCCTCACCCCTCGCCACCTTTCACCCTCATGAAAACACACATACACGCAAATACACTCCAGTTTTTGTTTTTGCATGAAATTGTATCTCAGTCTAAGGTCTCATGCTGTTGCTGCTACTGTCTTACTATTA.

Amino acid sequences are also known in the art. See, for example,UniProtKB/Swiss-Prot accession no. Q9Y243 (Akt3_HUMAN), which isspecifically incorporated by reference in its entirety and provides theamino acid sequence:

(SEQ ID NO: 2) MSDVTIVKEGWVQKRGEYIKNWRPRYFLLKTDGSFIGYKEKPQDVDLPYPLNNFSVAKCQLMKTERPKPNTFIIRCLQWTTVIERTFHVDTPEEREEWTEATQAVADRLQRQEEERMNCSPTSQIDNIGEEEMDASTTHHKRKTMNDFDYLKLLGKGTFGKVILVREKASGKYYAMKILKKEVIIAKDEVAHTLTESRVLKNTRHPFLTSLKYSFQTKDRLCFVMEYVNGGELFFHLSRERVFSEDRTRFYGAEIVSALDYLHSGKIVYRDLKLENLMLDKDGHIKITDFGLCKEGITDAATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEDIKFPRTLSSDAKSLLSGLLIKDPNKRLGGGPDDAKEIMRHSFESGVNWQDVYDKKLVPPFKPQVTSETDTRYFDEEFTAQTITITPPEKYDEDGMDCMDNERRPHFPQFSYSASGRE.

The domain structure of Akt3 is reviewed in Romano, Scientifica, Volume2013 (2013), Article ID 317186, 12 pages, and includes an N-terminalpleckstrin homology domain (PH), followed by a catalytic kinase domain(KD), and the C-terminal regulatory hydrophobic region. The catalyticand regulatory domains are both important for the biological actionsmediated by Akt protein kinases and exhibit the maximum degree ofhomology among the three Akt isoforms. The PH domain binds lipidsubstrates, such as phosphatidylinositol (3,4) diphosphate (PIP2) andphosphatidylinositol (3,4,5) triphosphate (PIP3). The ATP binding siteis situated approximately in the middle of the catalytic kinase domain,which has a substantial degree of homology with the other components ofthe AGCkinases family, such as p70 S6 kinase (S6K) and p90 ribosomal S6kinase (RSK), protein kinase A (PKA) and protein kinase B (PKB). Thehydrophobic regulatory moiety is a typical feature of the AGC kinasesfamily. With reference to SEQ ID NO:2, Akt 3 is generally considered tohave the following molecule processing and domain structure outlinedbelow.

Molecule Processing:

Feature key Position(s) Length Description Initiator methionine 1 1Removed Chain 2-479 478 Akt3

Regions:

Feature key Position(s) Length Description Domain  5-107 103 PH Domain148-405 258 Protein kinase Domain 406-479 74 AGC-kinase C-terminalNucleotide binding 154-162 9 ATP

Sites:

Feature key Position(s) Length Description Active site 271 1 Protonacceptor Binding site 177 1 ATP

The initiator methionine of SEQ ID NO:2 is disposable for Akt3 function.Therefore, in some embodiments, the compound directly or indirectlyinhibits expression or bioavailability of an Akt3 having the amino acidsequence

(SEQ ID NO: 3) SDVTIVKEGWVQKRGEYIKNWRPRYFLLKTDGSFIGYKEKPQDVDLPYPLNNFSVAKCQLMKTERPKPNTFIIRCLQWTTVIERTFHVDTPEEREEWTEATQAVADRLQRQEEERMNCSPTSQIDNIGEEEMDASTTHHKRKTMNDFDYLKLLGKGTFGKVILVREKASGKYYAMKILKKEVIIAKDEVAHTLTESRVLKNTRHPFLTSLKYSFQTKDRLCFVMEYVNGGELFFHLSRERVFSEDRTRFYGAEIVSALDYLHSGKIVYRDLKLENLMLDKDGHIKITDFGLCKEGITDAATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEDIKFPRTLSSDAKSLLSGLLIKDPNKRLGGGPDDAKEIMRHSFESGVNWQDVYDKKLVPPFKPQVTSETDTRYFDEEFTAQTITITPPEKYDEDGMDCMDNERRPHFPQFSYSASGRE.

Two specific sites, one in the kinase domain (Thr-305 with reference toSEQ ID NO:2) and the other in the C-terminal regulatory region (Ser-472with reference to SEQ ID NO:2), need to be phosphorylated for fullactivation of Akt3. Interaction between the PH domain of Akt3 and TCL1Aenhances Akt3 phosphorylation and activation. IGF-1 leads to theactivation of Akt3, which may play a role in regulating cell survival.

Compounds 1-28 can inhibit Akt3 activity by binding to one or moreactive sites on the Akt3 polypeptide. A preferred binding site is one orboth of the kinase domains.

C. Formulations

Formulations of and pharmaceutical compositions including one or more ofcompounds 1-28 are provided. Generally, dosage levels, for the compoundsdisclosed herein are between about 0.0001 mg/kg of body weight to about1,000 mg/kg, more preferably of 0.001 to 500 mg/kg, more preferably 0.01to 50 mg/kg of body weight daily are administered to mammals.

1. Delivery Vehicles

Compounds 1-28 can be administered to a subject, preferably a humansubject, where it is taken up into the cells of a subject with orwithout the aid of a delivery vehicle. Appropriate delivery vehicles forthe disclosed active agents are known in the art and can be selected tosuit the particular active agent. For example, in some embodiments, thecompound is incorporated into or encapsulated by a nanoparticle,microparticle, micelle, synthetic lipoprotein particle, or carbonnanotube. For example, the compositions can be incorporated into avehicle such as polymeric microparticles which provide controlledrelease of the active agent(s). In some embodiments, release of thedrug(s) is controlled by diffusion of the active agent(s) out of themicroparticles and/or degradation of the polymeric particles byhydrolysis and/or enzymatic degradation. Suitable polymers includeethylcellulose and other natural or synthetic cellulose derivatives.Polymers which are slowly soluble and form a gel in an aqueousenvironment, such as hydroxypropyl methylcellulose or polyethylene oxidemay also be suitable as materials for drug containing microparticles.Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA),polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),poly-3-hydroxybut rate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof. In some embodiments,both agents are incorporated into the same particles and are formulatedfor release at different times and/or over different time periods. Forexample, in some embodiments, one of the agents is released entirelyfrom the particles before release of the second agent begins. In otherembodiments, release of the first agent begins followed by release ofthe second agent before the all of the first agent is released. In stillother embodiments, both agents are released at the same time over thesame period of time or over different periods of time.

The compounds can be incorporated into a delivery vehicle prepared frommaterials which are insoluble in aqueous solution or slowly soluble inaqueous solution, but are capable of degrading within the GI tract bymeans including enzymatic degradation, surfactant action of bile acids,and/or mechanical erosion. As used herein, the term “slowly soluble inwater” refers to materials that are not dissolved in water within aperiod of 30 minutes. Preferred examples include fats, fatty substances,waxes, wax-like substances and mixtures thereof. Suitable fats and fattysubstances include fatty alcohols (such as lauryl, myristyl stearyl,cetyl or cetostearyl alcohol), fatty acids and derivatives, including,but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and tri-glycerides), and hydrogenated fats. Specific examples include,but are not limited to hydrogenated vegetable oil, hydrogenatedcottonseed oil, hydrogenated castor oil, hydrogenated oils availableunder the trade name Sterotex®, stearic acid, cocoa butter, and stearylalcohol. Suitable waxes and wax-like materials include natural orsynthetic waxes, hydrocarbons, and normal waxes.

Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C. Therelease point and/or period of release can be varied as discussed above.

2. Pharmaceutical Compositions

Pharmaceutical compositions including the disclosed compounds, with orwithout a delivery vehicle, are provided. Pharmaceutical compositionscan be formulated for administration by parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), enteral,transmucosal (nasal, vaginal, rectal, or sublingual), or transdermal(either passively or using iontophoresis or electroporation) routes ofadministration or using bioerodible inserts and can be formulated indosage forms appropriate for each route of administration.

In certain embodiments, the compositions are administered locally, forexample by injection directly into a site to be treated (e.g., into atumor). In some embodiments, the compositions are injected or otherwiseadministered directly into the vasculature onto vascular tissue at oradjacent to the intended site of treatment (e.g., adjacent to a tumor).Typically, local administration causes an increased localizedconcentration of the composition which is greater than that which can beachieved by systemic administration.

a. Formulations for Parenteral Administration

Compounds and pharmaceutical compositions thereof can be administered inan aqueous solution, by parenteral injection. The formulation may alsobe in the form of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of the activeagent(s) and optionally include pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions include diluents sterile water, buffered saline ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength; and optionally, additives such as detergents andsolubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to aspolysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

b. Enteral Formulations

Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art. Formulations may be prepared using apharmaceutically acceptable carrier. As generally used herein “carrier”includes, but is not limited to, diluents, preservatives, binders,lubricants, disintegrators, swelling agents, fillers, stabilizers, andcombinations thereof.

Carrier also includes all components of the coating composition, whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release dosage formulations may be prepared asdescribed in standard references. These references provide informationon carriers, materials, equipment and process for preparing tablets andcapsules and delayed release dosage forms of tablets, capsules, andgranules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name Eudragit®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients include, but are notlimited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactions,which include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can for formulated for controlled release. For example, theone or more compounds and optional one or more additional active agentscan be formulated into nanoparticles, microparticles, and combinationsthereof, and encapsulated in a soft or hard gelatin or non-gelatincapsule or dispersed in a dispersing medium to form an oral suspensionor syrup. The particles can be formed of the drug and a controlledrelease polymer or matrix. Alternatively, the drug particles can becoated with one or more controlled release coatings prior toincorporation in to the finished dosage form.

In another embodiment, the one or more compounds and optional one ormore additional active agents are dispersed in a matrix material, whichgels or emulsifies upon contact with an aqueous medium, such asphysiological fluids. In the case of gels, the matrix swells entrappingthe active agents, which are released slowly over time by diffusionand/or degradation of the matrix material. Such matrices can beformulated as tablets or as fill materials for hard and soft capsules.

In still another embodiment, the one or more compounds, and optional oneor more additional active agents are formulated into a sold oral dosageform, such as a tablet or capsule, and the solid dosage form is coatedwith one or more controlled release coatings, such as a delayed releasecoatings or extended release coatings. The coating or coatings may alsocontain the compounds and/or additional active agents.

Extended Release Dosage Forms

The extended release formulations are generally prepared as diffusion orosmotic systems, which are known in the art. A diffusion systemtypically consists of two types of devices, a reservoir and a matrix,and is well known and described in the art. The matrix devices aregenerally prepared by compressing the drug with a slowly dissolvingpolymer carrier into a tablet form. The three major types of materialsused in the preparation of matrix devices are insoluble plastics,hydrophilic polymers, and fatty compounds. Plastic matrices include, butare not limited to, methyl acrylate-methyl methacrylate, polyvinylchloride, and polyethylene. Hydrophilic polymers include, but are notlimited to, cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers. In certain preferred embodiments, theacrylic polymer is comprised of one or more ammonio methacrylatecopolymers. Ammonio methacrylate copolymers are well known in the art,and are described in NF XVII as fully polymerized copolymers of acrylicand methacrylic acid esters with a low content of quaternary ammoniumgroups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename Eudragit®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the tradenames Eudragit®RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D and Eudragit®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEudragit® RL30D and 1:40 in Eudragit® RS30D. The mean molecular weightis about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred.The code designations RL (high permeability) and RS (low permeability)refer to the permeability properties of these agents. Eudragit® RL/RSmixtures are insoluble in water and in digestive fluids. However,multiparticulate systems formed to include the same are swellable andpermeable in aqueous solutions and digestive fluids. The polymersdescribed above such as Eudragit® RL/RS may be mixed together in anydesired ratio in order to ultimately obtain a sustained-releaseformulation having a desirable dissolution profile. Desirablesustained-release multiparticulate systems may be obtained, forinstance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit® RS,and 10% Eudragit® RL and 90% Eudragit® RS. One skilled in the art willrecognize that other acrylic polymers may also be used, such as, forexample, Eudragit® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules, etc. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray—congealedor congealed and screened and processed.

Delayed Release Dosage Forms

Delayed release formulations can be created by coating a solid dosageform with a polymer film, which is insoluble in the acidic environmentof the stomach, and soluble in the neutral environment of the smallintestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit® (RohmPharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55(soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 andabove), Eudragit® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and Eudragits® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

c. Formulations for Pulmonary and Mucosal Administration

Active agent(s) and compositions thereof can be applied formulated forpulmonary or mucosal administration. The administration can includedelivery of the composition to the lungs, nasal, oral (sublingual,buccal), vaginal, or rectal mucosa.

In one embodiment, the compounds are formulated for pulmonary delivery,such as intranasal administration or oral inhalation. The respiratorytract is the structure involved in the exchange of gases between theatmosphere and the blood stream. The lungs are branching structuresultimately ending with the alveoli where the exchange of gases occurs.The alveolar surface area is the largest in the respiratory system andis where drug absorption occurs. The alveoli are covered by a thinepithelium without cilia or a mucus blanket and secrete surfactantphospholipids. The respiratory tract encompasses the upper airways,including the oropharynx and larynx, followed by the lower airways,which include the trachea followed by bifurcations into the bronchi andbronchioli. The upper and lower airways are called the conductingairways. The terminal bronchioli then divide into respiratorybronchiole, which then lead to the ultimate respiratory zone, thealveoli, or deep lung. The deep lung, or alveoli, is the primary targetof inhaled therapeutic aerosols for systemic drug delivery.

Pulmonary administration of therapeutic compositions comprised of lowmolecular weight drugs has been observed, for example, beta-androgenicantagonists to treat asthma. Other therapeutic agents that are active inthe lungs have been administered systemically and targeted via pulmonaryabsorption. Nasal delivery is considered to be a promising technique foradministration of therapeutics for the following reasons: the nose has alarge surface area available for drug absorption due to the coverage ofthe epithelial surface by numerous microvilli, the subepithelial layeris highly vascularized, the venous blood from the nose passes directlyinto the systemic circulation and therefore avoids the loss of drug byfirst-pass metabolism in the liver, it offers lower doses, more rapidattainment of therapeutic blood levels, quicker onset of pharmacologicalactivity, fewer side effects, high total blood flow per cm³, porousendothelial basement membrane, and it is easily accessible.

The term aerosol as used herein refers to any preparation of a fine mistof particles, which can be in solution or a suspension, whether or notit is produced using a propellant. Aerosols can be produced usingstandard techniques, such as ultrasonication or high-pressure treatment.

Carriers for pulmonary formulations can be divided into those for drypowder formulations and for administration as solutions. Aerosols forthe delivery of therapeutic agents to the respiratory tract are known inthe art. For administration via the upper respiratory tract, theformulation can be formulated into a solution, e.g., water or isotonicsaline, buffered or un-buffered, or as a suspension, for intranasaladministration as drops or as a spray. Preferably, such solutions orsuspensions are isotonic relative to nasal secretions and of about thesame pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0to pH 7.0. Buffers should be physiologically compatible and include,simply by way of example, phosphate buffers. For example, arepresentative nasal decongestant is described as being buffered to a pHof about 6.2. One skilled in the art can readily determine a suitablesaline content and pH for an innocuous aqueous solution for nasal and/orupper respiratory administration.

Preferably, the aqueous solution is water, physiologically acceptableaqueous solutions containing salts and/or buffers, such as phosphatebuffered saline (PBS), or any other aqueous solution acceptable foradministration to an animal or human. Such solutions are well known to aperson skilled in the art and include, but are not limited to, distilledwater, de-ionized water, pure or ultrapure water, saline,phosphate-buffered saline (PBS). Other suitable aqueous vehiclesinclude, but are not limited to, Ringer's solution and isotonic sodiumchloride. Aqueous suspensions may include suspending agents such ascellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gumtragacanth, and a wetting agent such as lecithin. Suitable preservativesfor aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

In another embodiment, solvents that are low toxicity organic (i.e.nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethylacetate, tetrahydrofuran, ethyl ether, and propanol may be used for theformulations. The solvent is selected based on its ability to readilyaerosolize the formulation. The solvent should not detrimentally reactwith the compounds. An appropriate solvent should be used that dissolvesthe compounds or forms a suspension of the compounds. The solvent shouldbe sufficiently volatile to enable formation of an aerosol of thesolution or suspension. Additional solvents or aerosolizing agents, suchas freons, can be added as desired to increase the volatility of thesolution or suspension.

In one embodiment, compositions may contain minor amounts of polymers,surfactants, or other excipients well known to those of the art. In thiscontext, “minor amounts” means no excipients are present that mightaffect or mediate uptake of the compounds in the lungs and that theexcipients that are present are present in amount that do not adverselyaffect uptake of compounds in the lungs.

Dry lipid powders can be directly dispersed in ethanol because of theirhydrophobic character. For lipids stored in organic solvents such aschloroform, the desired quantity of solution is placed in a vial, andthe chloroform is evaporated under a stream of nitrogen to form a drythin film on the surface of a glass vial. The film swells easily whenreconstituted with ethanol. To fully disperse the lipid molecules in theorganic solvent, the suspension is sonicated. Nonaqueous suspensions oflipids can also be prepared in absolute ethanol using a reusable PARI LCJet+ nebulizer (PARI Respiratory Equipment, Monterey, Calif.).

Dry powder formulations (“DPFs”) with large particle size have improvedflowability characteristics, such as less aggregation, easieraerosolization, and potentially less phagocytosis. Dry powder aerosolsfor inhalation therapy are generally produced with mean diametersprimarily in the range of less than 5 microns, although a preferredrange is between one and ten microns in aerodynamic diameter. Large“carrier” particles (containing no drug) have been co-delivered withtherapeutic aerosols to aid in achieving efficient aerosolization amongother possible benefits.

Polymeric particles may be prepared using single and double emulsionsolvent evaporation, spray drying, solvent extraction, solventevaporation, phase separation, simple and complex coacervation,interfacial polymerization, and other methods well known to those ofordinary skill in the art. Particles may be made using methods formaking microspheres or microcapsules known in the art. The preferredmethods of manufacture are by spray drying and freeze drying, whichentails using a solution containing the surfactant, spraying to formdroplets of the desired size, and removing the solvent.

The particles may be fabricated with the appropriate material, surfaceroughness, diameter and tap density for localized delivery to selectedregions of the respiratory tract such as the deep lung or upper airways.For example, higher density or larger particles may be used for upperairway delivery. Similarly, a mixture of different sized particles,provided with the same or different EGS may be administered to targetdifferent regions of the lung in one administration.

Formulations for pulmonary delivery include unilamellar phospholipidvesicles, liposomes, or lipoprotein particles. Formulations and methodsof making such formulations containing nucleic acid are well known toone of ordinary skill in the art. Liposomes are formed from commerciallyavailable phospholipids supplied by a variety of vendors includingAvanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, theliposome can include a ligand molecule specific for a receptor on thesurface of the target cell to direct the liposome to the target cell.

d. Transdermal

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations can includepenetration enhancers.

III. Methods of Selectively Inhibiting Akt3

The disclosed compositions for selectively inhibiting Akt3 can be usedto modulate an immune response decreasing a suppressive function ofnTregs. In some embodiments, the compositions are administeredsystemically. In other embodiments, the compositions are administeredlocally or regionally. For example, in some embodiments, compositionscontaining one or more of compounds 1-28 are delivered to orspecifically target the tissue or organs in need of modulation. Tregscan be modulated by targeting or delivering the compositions to thelymph nodes. nTregs can be modulated by targeting or specificallydelivering the compositions to the thymus or spleen. iTregs can bemodulated by targeting or specifically delivering the compositions toconventional T cells outside the thymus.

In some in vivo approaches, the compositions disclosed herein areadministered to a subject in a therapeutically effective amount. As usedherein the term “effective amount” or “therapeutically effective amount”means a dosage sufficient to treat, inhibit, or alleviate one or moresymptoms of the disorder being treated or to otherwise provide a desiredpharmacologic and/or physiologic effect. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease, and the treatmentbeing effected. Exemplary symptoms, pharmacologic, and physiologiceffects are discussed in more detail below.

In some embodiments, the effect of the composition on a subject iscompared to a control. For example, the effect of the composition on aparticular symptom, pharmacologic, or physiologic indicator can becompared to an untreated subject, or the condition of the subject priorto treatment. In some embodiments, the symptom, pharmacologic, orphysiologic indicator is measured in a subject prior to treatment, andagain one or more times after treatment is initiated. In someembodiments, the control is a reference level, or average determinedbased measuring the symptom, pharmacologic, or physiologic indicator inone or more subjects that do not have the disease or condition to betreated (e.g., healthy subjects). In some embodiments, the effect of thetreatment is compared to a conventional treatment that is known the art.For example, if the disease to be treated is cancer, and conventionaltreatment could a chemotherapeutic agent.

In some embodiments, the immune modulating compositions disclosed hereinare administered in combination with one or more additional activeagents. The combination therapies can include administration of theactive agents together in the same admixture, or in separate admixtures.Therefore, in some embodiments, the pharmaceutical composition includestwo, three, or more active agents. The pharmaceutical compositions canbe formulated as a pharmaceutical dosage unit, referred to as a unitdosage form. Such formulations typically include an effective amount ofone or more of the disclosed immune modulating compounds. The differentactive agents can have the same, or different mechanisms of action. Insome embodiments, the combination results in an additive effect on thetreatment of the disease or disorder. In some embodiments, thecombinations result in a more than additive effect on the treatment ofthe disease or disorder.

Preferably, the disclosed compounds and methods of use specificallyinhibit the activity of Akt3 without increasing or decreasing theactivity of Akt1, Akt2, or the combination thereof.

A. Decreasing Immune Suppressive Responses and Increasing ImmuneStimulatory Responses

1. Methods of Treatment

In some embodiments compositions that decrease the bioactivity of Akt3are administered to a subject in an effective amount to increase animmune stimulatory response, decrease an immune suppressive response, ora combination thereof. Akt3 regulates the function and induction ofnatural and induced Tregs. Therefore Akt3 expression levels can bemodulated to alter the function and induction of Tregs. In someembodiments, a composition that selectively inhibits Akt3 isadministered to a subject in an effective amount to decrease asuppressive function of nTreg, to decrease the induction of conventionalTreg into iTreg, or a combination thereof. In some embodiments, adecrease in the suppressive function of nTreg is measured as an overalldecrease in secretion or presence of pro-inflammatory cytokines orchemokines, for example, TGFβ and IL10. Other pro-inflammatory moleculesthat can be decreased include, but are not limited to, IL-1β, TNF-α,IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. Induction ofconventional Treg into iTreg can be measured as differentiation ofCD4+CD25− cells into Foxp3+ cells. In some embodiments, this is measuredas an increase in the number of CD4+ conventional T cells, or a decreasein the number of Foxp3+ T cells.

2. Diseases to Treat

Compositions containing one or more of compounds 1-28 that selectivelyinhibit Akt3 can be used to increase an immune stimulatory response insubject. In some embodiments, the subjects have cancer, an infectiousdisease, or another condition in which the immune response is desired.In some embodiments, the subject does not have cancer or does not havean infectious disease. In some embodiments, the subject has aninfectious disease, but does not have cancer. In some embodiments, thesubject has cancer, but does not have an infectious disease.

a. Cancer

Compounds 1-28 for selectively inhibiting Akt3 provided herein aregenerally useful in vivo and ex vivo as immune response-stimulatingtherapeutics. In general, the compounds 1-28 for selectively inhibitingAkt3 are useful for treating a subject having or being predisposed toany disease or disorder to which the subject's immune system mounts animmune response. The ability of compounds to inhibit Akt3 and therebyinhibit or reduce Treg mediated immune suppression enables a more robustimmune response to be possible. The disclosed compositions are useful tostimulate or enhance immune stimulating or activating responsesinvolving T cells.

Compounds 1-28 are useful for stimulating or enhancing an immuneresponse in a host for treating cancer by selectively inhibiting Akt3.The compounds can be administered to a subject in an amount effective tostimulate T cells in the subject. The types of cancer that can betreated with the provided compositions and methods include, but are notlimited to, the following: bladder, brain, breast, cervical,colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,pancreatic, prostate, skin, stomach, uterine, ovarian, testicular andhematologic.

Malignant tumors that can be treated can be classified herein accordingto the embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands.Sarcomas, which arise less frequently, are derived from mesodermalconnective tissues such as bone, fat, and cartilage. The leukemias andlymphomas are malignant tumors of hematopoietic cells of the bonemarrow. Leukemias proliferate as single cells, whereas lymphomas tend togrow as tumor masses. Malignant tumors may show up at numerous organs ortissues of the body to establish a cancer.

b. Infections

Compounds 1-28 are generally useful in vivo and ex vivo as immuneresponse-stimulating therapeutics. In a preferred embodiment, thecompositions are useful for treating infections in which T cellexhaustion or T cell anergy has occurred causing the infection to remainwith the host over a prolonged period of time. Exemplary infections tobe treated are chronic infections cause by a hepatitis virus, a humanimmunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), aherpes virus, an Epstein-Barr virus, or a human papilloma virus. It willbe appreciated that other infections can also be treated using thecompounds for decreasing the bioavailability of Akt3. The disclosedcompositions are also useful as part of a vaccine. In a preferredembodiment, the type of disease to be treated or prevented is a chronicinfectious disease caused by a bacterium, virus, protozoan, helminth, orother microbial pathogen that enters intracellularly and is attacked,i.e., by cytotoxic T lymphocytes.

Chronic infections in human and animal models are associated with afailure of the host immune response to generate and sustain functionalCD8⁺ and CD4⁺ T-cell populations, which also results in poor antibodyresponses to neutralize infectivity. This loss of function is referredto as T cell exhaustion. T cell anergy is a tolerance mechanism in whichthe lymphocyte is intrinsically functionally inactivated following anantigen encounter, but remains alive for an extended period of time in ahyporesponsive state. One method for treating chronic infection is torevitalize exhausted T cells or to reverse T cell exhaustion in asubject as well as overcoming T cell anergy. Therefore, in someembodiments, compounds 1-28 for selectively inhibiting Akt3 areadministered to a subject in an effective amount to reverse T cellexhaustion, overcoming T cell anergy, or a combination thereof in asubject in need thereof.

Because viral infections are cleared primarily by T-cells, an increasein T-cell activity is therapeutically useful in situations where morerapid or thorough clearance of an infective viral agent would bebeneficial to an animal or human subject. Thus, the compounds can beadministered for the treatment of local or systemic viral infections,including, but not limited to, immunodeficiency (e.g., HIV), papilloma(e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., humaninfluenza virus A), and common cold (e.g., human rhinovirus) viralinfections. For example, pharmaceutical formulations including thecompounds can be administered topically to treat viral skin diseasessuch as herpes lesions or shingles, or genital warts. Pharmaceuticalformulations containing a compound for decreasing the bioavailability ofAkt3 can also be administered to treat systemic viral diseases,including, but not limited to, AIDS, influenza, the common cold, orencephalitis.

Representative infections that can be treated, include but are notlimited to infections cause by microoganisms including, but not limitedto, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Histoplasma, Hyphomicrobium,Legionella, Leishmania, Leptspirosis, Listeria, Meningococcus A, B andC, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus,Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia,Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Plasmodium vivax, Trypanosomabrucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalisand Schistosoma mansoni.

3. Use of Compounds for Selective Inhibition of Akt3 in Vaccines

a. Vaccine-Related Methods

Compounds 1-28 that selectively inhibit Akt3 can be administered aloneor in combination with any other suitable treatment. In one embodimentone or more of the compounds can be administered in conjunction with, oras a component of a vaccine composition. The disclosed compounds can beadministered prior to, concurrently with, or after the administration ofa vaccine. In one embodiment the compound is administered at the sametime as administration of a vaccine.

Compounds 1-28 for selectively inhibiting Akt3 can be administered inconjunction with prophylactic vaccines, which confer resistance in asubject to subsequent exposure to infectious agents, or in conjunctionwith therapeutic vaccines, which can be used to initiate or enhance asubject's immune response to a pre-existing antigen, such as a viralantigen in a subject infected with a virus.

The desired outcome of a prophylactic, therapeutic or de-sensitizedimmune response may vary according to the disease, according toprinciples well known in the art. For example, an immune responseagainst an infectious agent may completely prevent colonization andreplication of an infectious agent, affecting “sterile immunity” and theabsence of any disease symptoms. However, a vaccine against infectiousagents may be considered effective if it reduces the number, severity orduration of symptoms; if it reduces the number of individuals in apopulation with symptoms; or reduces the transmission of an infectiousagent. Similarly, immune responses against cancer, allergens orinfectious agents may completely treat a disease, may alleviatesymptoms, or may be one facet in an overall therapeutic interventionagainst a disease.

The compounds induce an improved effector cell response such as a CD4T-cell immune response, against at least one of the component antigen(s)or antigenic compositions compared to the effector cell responseobtained with the corresponding composition without the compound. Theterm “improved effector cell response” refers to a higher effector cellresponse such as a CD4 T cell response obtained in a human patient afteradministration of the vaccine composition than that obtained afteradministration of the same composition without a compound for decreasingthe bioavailability of Akt3. Such a formulation can advantageously beused to induce anti-antigen effector cell response capable of detectionof antigen epitopes presented by MHC class II molecules.

The improved effector cell response can be obtained in animmunologically unprimed patient, i.e. a patient who is seronegative tothe antigen. This seronegativity may be the result of the patient havingnever faced the antigen (so-called “naïve” patient) or, alternatively,having failed to respond to the antigen once encountered. Preferably theimproved effector cell response is obtained in an immunocompromisedsubject such as an elderly, typically 65 years of age or above, or anadult younger than 65 years of age with a high risk medical condition(“high risk” adult), or a child under the age of two.

The improved effector cell response can be assessed by measuring thenumber of cells producing any of the following cytokines: (1) cellsproducing at least two different cytokines (CD40L, IL-2, IFNγ, TNF-α,IL-17); (2) cells producing at least CD40L and another cytokine (IL-2,TNF-α, IFNγ, IL-17); (3) cells producing at least IL-2 and anothercytokine (CD40L, TNF-alpha, IFNγ, IL-17); (4) cells producing at leastIFNγ and another cytokine (IL-2, TNF-α, CD40L, IL-17); (5) cellsproducing at least TNF-α and another cytokine (IL-2, CD40L, IFNγ,IL-17); and (6) cells producing at least IL-17 and another cytokine(TNF-alpha, IL-2, CD40L, IFNγ, IL-17)

An improved effector cell response is present when cells producing anyof the above cytokines will be in a higher amount followingadministration of the vaccine composition compared to the administrationof the composition without a compound for decreasing the bioavailabilityof Akt3. Typically at least one, preferably two of the five conditionsmentioned above will be fulfilled. In a preferred embodiment, cellsproducing all five cytokines (CD40L, IL-2, IFNγ, TNF-α, IL-17) will bepresent at a higher number in the vaccinated group compared to theun-vaccinated group.

The immunogenic compositions may be administered by any suitabledelivery route, such as intradermal, mucosal e.g. intranasal, oral,intramuscular or subcutaneous. Other delivery routes are well known inthe art. The intramuscular delivery route is preferred for theimmunogenic compositions. Intradermal delivery is another suitableroute. Any suitable device may be used for intradermal delivery, forexample short needle devices. Intradermal vaccines may also beadministered by devices which limit the effective penetration length ofa needle into the skin. Jet injection devices which deliver liquidvaccines to the dermis via a liquid jet injector or via a needle whichpierces the stratum corneum and produces a jet which reaches the dermiscan also be used. Jet injection devices are known in the art. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis can also be used. Additionally, conventional syringes can be usedin the classical Mantoux method of intradermal administration.

Another suitable administration route is the subcutaneous route. Anysuitable device may be used for subcutaneous delivery, for exampleclassical needle. Preferably, a needle-free jet injector service isused. Needle-free injectors are known in the art. More preferably thedevice is pre-filled with the liquid vaccine formulation.

Alternatively the vaccine is administered intranasally. Typically, thevaccine is administered locally to the nasopharyngeal area, preferablywithout being inhaled into the lungs. It is desirable to use anintranasal delivery device which delivers the vaccine formulation to thenasopharyngeal area, without or substantially without it entering thelungs. Preferred devices for intranasal administration of the vaccinesare spray devices. Nasal spray devices are commercially available.Nebulizers produce a very fine spray which can be easily inhaled intothe lungs and therefore does not efficiently reach the nasal mucosa.Nebulizers are therefore not preferred. Preferred spray devices forintranasal use are devices for which the performance of the device isnot dependent upon the pressure applied by the user. These devices areknown as pressure threshold devices. Liquid is released from the nozzleonly when a threshold pressure is applied. These devices make it easierto achieve a spray with a regular droplet size. Pressure thresholddevices suitable for use with the present invention are known in the artand are commercially available.

Preferred intranasal devices produce droplets (measured using water asthe liquid) in the range 1 to 200 μm, preferably 10 to 120 μm. Below 10μm there is a risk of inhalation, therefore it is desirable to have nomore than about 5% of droplets below 10 μm. Droplets above 120 μm do notspread as well as smaller droplets, so it is desirable to have no morethan about 5% of droplets exceeding 120 μm.

Bi-dose delivery is another feature of an intranasal delivery system foruse with the vaccines. Bi-dose devices contain two sub-doses of a singlevaccine dose, one sub-dose for administration to each nostril.Generally, the two sub-doses are present in a single chamber and theconstruction of the device allows the efficient delivery of a singlesub-dose at a time. Alternatively, a monodose device may be used foradministering the vaccines.

The immunogenic composition may be given in two or more doses, over atime period of a few days, weeks or months. In one embodiment, differentroutes of administration are utilized, for example, for the firstadministration may be given intramuscularly, and the boostingcomposition can be administered through a different route, for exampleintradermal, subcutaneous or intranasal.

The improved effector cell response conferred by the immunogeniccomposition may be ideally obtained after one single administration. Thesingle dose approach is extremely relevant in a rapidly evolvingoutbreak situation including bioterrorist attacks and epidemics. Incertain circumstances, especially for the elderly population, or in thecase of young children (below 9 years of age) who are vaccinated for thefirst time against a particular antigen, it may be beneficial toadminister two doses of the same composition. The second dose of thesame composition (still considered as ‘composition for firstvaccination’) can be administered during the on-going primary immuneresponse and is adequately spaced in time from the first dose. Typicallythe second dose of the composition is given a few weeks, or about onemonth, e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after thefirst dose, to help prime the immune system in unresponsive or poorlyresponsive individuals.

In a specific embodiment, the administration of the immunogeniccomposition alternatively or additionally induces an improved B-memorycell response in patients administered with the adjuvanted immunogeniccomposition compared to the B-memory cell response induced inindividuals immunized with the un-adjuvanted composition. An improvedB-memory cell response is intended to mean an increased frequency ofperipheral blood B lymphocytes capable of differentiation intoantibody-secreting plasma cells upon antigen encounter as measured bystimulation of in vitro differentiation (see Example sections, e.g.methods of Elispot B cells memory).

In a still another embodiment, the immunogenic composition increases theprimary immune response as well as the CD8 T cell response. Theadministration of a single dose of the immunogenic composition for firstvaccination provides better sero-protection and induces an improved CD4T-cell, or CD8 T-cell immune response against a specific antigencompared to that obtained with the un-adjuvanted formulation. This mayresult in reducing the overall morbidity and mortality rate andpreventing emergency admissions to hospital for pneumonia and otherinfluenza-like illness. This method allows inducing a CD4 T cellresponse which is more persistent in time, e.g. still present one yearafter the first vaccination, compared to the response induced with theun-adjuvanted formulation.

Preferably the CD4 T-cell immune response, such as the improved CD4T-cell immune response obtained in an unprimed subject, involves theinduction of a cross-reactive CD4 T helper response. In particular, theamount of cross-reactive CD4 T cells is increased. The term“cross-reactive” CD4 response refers to CD4 T-cell targeting sharedepitopes for example between influenza strains.

b. Immunogenic and Vaccine Compositions

The dose of compounds 1-28 for selectively inhibiting Akt3 enhances animmune response to an antigen in a human. As discussed above, compoundsfor decreasing the bioavailability of Akt3 described herein can beadministered as a component of a vaccine to promote, augment, or enhancethe primary immune response and effector cell activity and numbers. Whenused as part of a vaccine, the compound can be administered in separate,or in the same admixture with an immunogenic composition or as part ofan immunogenic protocol. Vaccines include antigens, and optionally otheradjuvants and targeting molecules.

i. Antigens

Antigens can be peptides, proteins, polysaccharides, saccharides,lipids, nucleic acids, or combinations thereof. The antigen can bederived from a virus, bacterium, parasite, protozoan, fungus,histoplasma, tissue or transformed cell and can be a whole cell orimmunogenic component thereof, e.g., cell wall components or molecularcomponents thereof.

Suitable antigens are known in the art and are available fromcommercial, government and scientific sources. In one embodiment, theantigens are whole inactivated or attenuated organisms. These organismsmay be infectious organisms, such as viruses, parasites and bacteria.The antigens may be tumor cells or cells infected with a virus orintracellular pathogen such as gonorrhea or malaria. The antigens may bepurified or partially purified polypeptides derived from tumors or viralor bacterial sources. The antigens can be recombinant polypeptidesproduced by expressing DNA encoding the polypeptide antigen in aheterologous expression system. The antigens can be DNA encoding all orpart of an antigenic protein. The DNA may be in the form of vector DNAsuch as plasmid DNA.

Antigens may be provided as single antigens or may be provided incombination. Antigens may also be provided as complex mixtures ofpolypeptides or nucleic acids.

(a) Viral Antigens

A viral antigen can be isolated from any virus including, but notlimited to, a virus from any of the following viral families:Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus,Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus,Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acuterespiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae,Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virusand Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)),Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2,Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae(e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus),Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae,Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae,Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytialvirus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpoxvirus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus,such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae(for example, rabies virus, measles virus, respiratory syncytial virus,etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), andTotiviridae. Suitable viral antigens also include all or part of Dengueprotein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and DengueD1NS3.

Viral antigens may be derived from a particular strain, or a combinationof strains, such as a papilloma virus, a herpes virus, i.e. herpessimplex 1 and 2; a hepatitis virus, for example, hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), the tick-borne encephalitis viruses; parainfluenza,varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus,adenovirus, coxsackieviruses, equine encephalitis, Japaneseencephalitis, yellow fever, Rift Valley fever, and lymphocyticchoriomeningitis.

(b) Bacterial Antigens

Bacterial antigens can originate from any bacteria including, but notlimited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

(c) Parasitic Antigens

Antigens of parasites can be obtained from parasites such as, but notlimited to, antigens derived from Cryptococcus neoformans, Histoplasmacapsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides,Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoanantigens, Plasmodian antigens, such as all or part of a Circumsporozoiteprotein, a Sporozoite surface protein, a liver stage antigen, an apicalmembrane associated protein, or a Merozoite surface protein.

(d) Tumor Antigens

The antigen can be a tumor antigen, including a tumor-associated ortumor-specific antigen, such as, but not limited to, alpha-actinin-4,Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a,coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein,LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2,KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9,pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras,Bage-1, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, Mage-A1, 2, 3, 4,6, 10, 12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2,MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1,MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1,Horn/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,and TPS. Tumor antigens, such as BCG, may also be used as animmunostimulant to adjuvant.

ii. Adjuvants

Optionally, the vaccines may include an adjuvant. The adjuvant can be,but is not limited to, one or more of the following: oil emulsions(e.g., Freund's adjuvant); saponin formulations; virosomes andviral-like particles; bacterial and microbial derivatives;immunostimulatory oligonucleotides; ADP-ribosylating toxins anddetoxified derivatives; alum; BCG; mineral-containing compositions(e.g., mineral salts, such as aluminium salts and calcium salts,hydroxides, phosphates, sulfates, etc.); bioadhesives and/ormucoadhesives; microparticles; liposomes; polyoxyethylene ether andpolyoxyethylene ester formulations; polyphosphazene; muramyl peptides;imidazoquinolone compounds; and surface active substances (e.g.lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol).

Adjuvants may also include immunomodulators such as cytokines,interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-.gamma.), macrophage colony stimulatingfactor, and tumor necrosis factor. Other co-stimulatory molecules,including other polypeptides of the B7 family, may also be administered.Such proteinaceous adjuvants may be provided as the full-lengthpolypeptide or an active fragment thereof, or in the form of DNA, suchas plasmid DNA.

4. Combination Therapies

The disclosed compositions for selectively inhibiting Akt3 can beadministered alone or in combination with one, two, three, or moreadditional active agents. In some embodiments, the additional activeagent is one that is known in the art for treatment of cancer,infections, or administered in combination with a vaccine, etc. Theadditional therapeutic agents are selected based on the condition,disorder or disease to be treated. For example, compositions forselectively inhibiting Akt3 can be co-administered with one or moreadditional agents that function to enhance or promote an immuneresponse.

For example, the disclosed compositions can be administered with anantibody or antigen binding fragment thereof specific for a growthfactor receptors or tumor specific antigens. Representative growthfactors receptors include, but are not limited to, epidermal growthfactor receptor (EGFR; HER1); c-erbB2 (HER2); c-erbB3 (HER3); c-erbB4(HER4); insulin receptor; insulin-like growth factor receptor 1(IGF-1R); insulin-like growth factor receptor 2/Mannose-6-phosphatereceptor (IGF-II R/M-6-P receptor); insulin receptor related kinase(IRRK); platelet-derived growth factor receptor (PDGFR);colony-stimulating factor-1receptor (CSF-1R) (c-Fms); steel receptor(c-Kit); Flk2/Flt3; fibroblast growth factor receptor 1 (Flg/Cek1);fibroblast growth factor receptor 2 (Bek/Cek3/K-Sam); Fibroblast growthfactor receptor 3; Fibroblast growth factor eceptor 4; nerve growthfactor receptor (NGFR) (TrkA); BDNF receptor (TrkB); NT-3-receptor(TrkC); vascular endothelial growth factor receptor 1 (Flt1); vascularendothelial growth factor receptor 2/Flk1/KDR; hepatocyte growth factorreceptor (HGF-R/Met); Eph; Eck; Eek; Cek4/Mek4/HEK; Cek5; Elk/Cek6;Cek7; Sek/Cek8; Cek9; Cek10; HEK11; 9 Ror1; Ror2; Ret; Axl; RYK; DDR;and Tie.

Additional therapeutic agents include conventional cancer therapeuticssuch as chemotherapeutic agents, cytokines, chemokines, and radiationtherapy. The majority of chemotherapeutic drugs can be divided in to:alkylating agents, antimetabolites, anthracyclines, plant alkaloids,topoisomerase inhibitors, and other antitumour agents. All of thesedrugs affect cell division or DNA synthesis and function in some way.Additional therapeutics include monoclonal antibodies and tyrosinekinase inhibitors e.g. imatinib mesylate (GLEEVEC® or GLIVEC®), whichdirectly targets a molecular abnormality in certain types of cancer(chronic myelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited tocisplatin, carboplatin, doxorubicin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine,vindesine, taxol and derivatives thereof, irinotecan, topotecan,amsacrine, etoposide, etoposide phosphate, teniposide,epipodophyllotoxins, trastuzumab (HERCEPTIN®), cetuximab, and rituximab(RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinationsthereof.

In a preferred embodiment, the additional therapeutic agent iscyclophosphamide. Cyclophosphamide (CPA, Cytoxan, or Neosar) is anoxazahosphorine drug and analogs include ifosfamide (IFO, Ifex),perfosfamide, trophosphamide (trofosfamide; Ixoten), andpharmaceutically acceptable salts, solvates, prodrugs and metabolitesthereof (US patent application 20070202077 which is incorporated in itsentirety). Ifosfamide (MITOXANAO) is a structural analog ofcyclophosphamide and its mechanism of action is considered to beidentical or substantially similar to that of cyclophosphamide.Perfosfamide (4-hydroperoxycyclophosphamide) and trophosphamide are alsoalkylating agents, which are structurally related to cyclophosphamide.For example, perfosfamide alkylates DNA, thereby inhibiting DNAreplication and RNA and protein synthesis. New oxazaphosphorinesderivatives have been designed and evaluated with an attempt to improvethe selectivity and response with reduced host toxicity (Ref. Liang J,Huang M, Duan W, Yu X Q, Zhou S. Design of new oxazaphosphorineanticancer drugs. Curr Pharm Des. 2007; 13(9):963-78. Review). Theseinclude mafosfamide (NSC 345842), glufosfamide (D19575,beta-D-glucosylisophosphoramide mustard), S-(−)-bromofosfamide (CBM-11),NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060(aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorineanalog that is a chemically stable 4-thioethane sulfonic acid salt of4-hydroxy-CPA. Glufosfamide is IFO derivative in which theisophosphoramide mustard, the alkylating metabolite of IFO, isglycosidically linked to a beta-D-glucose molecule. Additionalcyclophosphamide analogs are described in U.S. Pat. No. 5,190,929entitled “Cyclophosphamide analogs useful as anti-tumor agents” which isincorporated herein by reference in its entirety.

Additional therapeutic agents include is an agent that reduces activityand/or number of regulatory T lymphocytes (T-regs), preferably Sunitinib(SUTENT®), or anti-TGFβ. Other additional therapeutic agents includemitosis inhibitors, such as paclitaxol, aromatase inhibitors (e.g.Letrozole), angiogenesis inhibitors (VEGF inhibitors e.g. Avastin,VEGF-Trap), TLR4 antagonists, and IL-18 antagonists.

V. Kits

Medical kits are also disclosed. The medical kits can include, forexample, a dosage supply of one or more of compounds 1-28 disclosedherein. The compounds can be supplied alone (e.g., lyophilized), or in apharmaceutical composition. The compounds can be in a unit dosage, or ina stock that should be diluted prior to administration. In someembodiments, the kit includes a supply of pharmaceutically acceptablecarrier. The kit can also include devices for administration of theactive agent(s) or composition(s), for example, syringes. The kits caninclude printed instructions for administering the compound in a use asdescribed above.

Examples Example 1:4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide(Formula 1) Inhibits Akt3 but not Akt1 Phosphorylation in Tregs

Materials and Methods

FACS-sorted natural regulatory T cells (nTregs), from WT C57BL/6J(foxp3-GFP) mice were plated on anti-CD3-coated plates and cultured inactivation media (IL2 and anti-CD28) without inhibitors (Stimulated) andwith different concentrations of inhibitor4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide(also referred to as JJ64-E) for 72 hrs. For negative control(Non-stimulated-NS) cells were left in media containing IL-2 for 72 hrs.nTreg cell lysates prepared on day 3 (72 hrs) of treatment wereseparated by SDS-PAGE and immunoblotted with specific antibodies (pAkt1)or pAkt3; actin was used as loading control.

Results

The data show4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamideinhibits Akt3 but not Akt1 phosphorylation in Tregs.

Example 2:4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide(Formula 1) Selectively Inhibits Treg Proliferation

Materials and Methods

FACS-sorted nTregs, CD4+ and CD8+ T cells from C57BL/6J(foxp3-GFP) wereplated on anti-CD3-coated plates and cultured in activation media (IL2and anti-CD28) without inhibitors (Stimulated) and with inhibitors(JJ64-E) for 72 hrs. For negative control (Non-stimulated-NS) cells wereleft in media containing IL-2 for 72 hrs. After 72 hrs Proliferation(level of VCT) in live gated cells was measured by flow cytometry.

Results

The data show that4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamideselectively inhibits Treg proliferation sparing CD8 and other CD4 Tcells.

Example 3:4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamideDecreases Tegs In Vivo in TC-1 Tumor Model

Materials and Methods

WT C57BL/6J mice (n=3/group) were injected s.c. in the right flank with7×10⁴ TC-1 cells. Mice from appropriate groups were treated with either5 mg/kg or 10 mg/kg of JJ64-E injected (i.p.) every day starting on day10 after tumor implantation throughout the experiment. All groups wereeuthanized on day 15 of TC-1 implantation. The percentage of Tregs(CD4+Foxp3+) was analyzed by flow cytometry.

Statistical significance was determined by one-way ANOVA with Tukey'smultiple comparison test (*, p<0.05; **, p<0.01; ***, p<0.001).

Results

4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamidedecreases Tegs in vivo in TC-1 tumor model

Example 4:4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide(Formula 1 or JJ64-E) does not Affect CD8 and Other (FoxP3neg) CD4 TCells in TC-1 Tumor Model

Materials and Methods

WT C57BL/6 4-6 weeks old female mice (n=5/group) were injected s.c. inthe right flank with 7×104 TC-1 cells. Mice from appropriate groups weretreated with either 5 mg/kg or 10 mg/kg of JJ64-E injected (i.p.)everyday starting on day 10 after tumor implantation throughout theexperiment. All groups were euthanized on day 15 of TC-1 implantation.The percentage of CD4 and CD8 were analyzed in splenic CD4+ cells byflow cytometry.

Results

4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamidedoes not affect CD8 and other (FoxP3neg) CD4 T cells in TC-1 tumormodel.

Example 5:4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamide(in Both Applications Formula 1 or JJ64-E) Inhibits TC-1 Tumor Growthand Prolongs the Survival at High Dose as Monotherapy and at Lower Dosewhen Combined with Vaccine

Materials and Methods

C57BL/6 mice (n=5/group) were injected s.c. in the right flank with7×10⁴ TC-1 cells. Mice from appropriate groups were injected weekly withvaccine (s.c.) or DMSO 5% as a control. Mice were also treated withvaccine (weekly) along with either 10 mg/kg or 20 mg/kg of JJ64-Einjected (i.p.) every day starting on day 6 after tumor implantationthroughout the experiment.

FIGS. 5B and 5C are bar diagrams showing average tumor volumes of micefor each group. FIG. 5D is a Kaplan-Meier plot of the overall survival.Statistical significance was determined by Log-rank (Mantel-Cox) test.

Statistical significance was determined by one-way ANOVA with Tukey'smultiple comparison test (*, p<0.05; **, p<0.01; ***, p<0.001).

Results

4-[(6-nitroquinolin-4-yl)amino]-N-[4-(pyridin-4-ylamino)phenyl]benzamideinhibits TC-1 tumor growth and prolongs the survival at high dose asmonotherapy and at lower dose when combined with vaccine.

Example 6: JJ64-B Modification (Formula 3) Inhibits iTreg Induction

Materials and Methods

FACS-sorted CD4+FoxP3− cells were plated on anti-CD3-coated plates withsoluble IL2 and ant-CD28 with TGF-β (induction) Cell were induced foriTregs with JJ64-B (FIG. 6A) without inhibitor (Induction-IND)) or for72 hrs. Cells were harvested and the frequency of CD4+FoxP3+ cells wasmeasured by flow cytometry.

Results

JJ64-B (FIG. 6A) inhibits iTreg induction.

Example 7: JJ64-C (FIG. 7A or Formula 18) Inhibits iTreg Induction

Materials and Methods

FACS-sorted CD4+FoxP3− cells were plated on anti-CD3-coated plates withsoluble IL2 and ant-CD28 with TGF-β (induction) Cell were induced foriTregs with JJ64-C (JJ64 modified drug C or Formula 18) or withoutinhibitor (Induction-IND)) or for 72 hrs. Cells were harvested and thefrequency of CD4+FoxP3+ cells was measured by flow cytometry.

Results

The data show that compound 18 (FIG. 7A) inhibits iTregs induction.

Example 8: JJ64-C (FIG. 7A or Formula 18) Inhibits TC-1 Tumor Growth andProlongs the Survival at High Dose as Monotherapy and at Lower Dose whenCombined with Vaccine

Materials and Methods

C57BL/6 mice (n=5/group) were injected s.c. in the right flank with7×10⁴ TC-1 cells. Mice from appropriate groups were injected weekly withvaccine (s.c.) or DMSO 5% as a control. Mice were also treated withvaccine (weekly) along with either 10 mg/kg or 20 mg/kg of JJ64-C (orFIG. 7A or Formula 18) injected (i.p.) every other day starting on day 6after tumor implantation throughout the experiment.

FIGS. 7C and 7D are bar diagrams representing average tumor volumes ofmice for each group. FIG. 7E is a Kaplan-Meier plot of the overallsurvival. Statistical significance was determined by Log-rank(Mantel-Cox) test.

Statistical significance was determined by one-way ANOVA with Tukey'smultiple comparison test (*, p<0.05; **, p<0.01; ***, p<0.001).

Results

JJ64-C (FIG. 7A or Formula 18) inhibits TC-1 tumor growth and prolongsthe survival at high dose as monotherapy and at lower dose when combinedwith vaccine.

Example 9: JJ64-D (FIG. 9A) Inhibits iTreg Induction

Materials and Methods

FACS-sorted CD4+FoxP3− cells were plated on anti-CD3-coated plates withsoluble IL2 and ant-CD28 with TGF-β (induction) Cell were induced foriTregs with JJ64-D (JJ64 modified drug D or FIG. 9A) or withoutinhibitor (Induction-IND)) or for 72 hrs. Cells were harvested and thefrequency of CD4+FoxP3+ cells was measured by flow cytometry.

Results

JJ64-D (FIG. 9A) inhibits iTreg induction.

We claim:
 1. A compound according to Formula I

or an enantiomer, polymorph, or pharmaceutically acceptable saltthereof.
 2. A pharmaceutical composition comprising the compound ofclaim 1.